The investigations in this proposal are focused on identifying the molecular basis for the direct and indirect consequences of Vitamin D Receptor (VDR) ablation. VDR knockout mice develop hypocalcemia and secondary hyperparathyroidism which, in turn, leads to hypophosphatemia. They also develop rickets and osteomalacia. However, when placed on a special diet that maintains normal mineral ion levels, the VDR null mice have histologically, histomorphometrically and biomechanically normal long bones, thus the skeletal effects of VDR ablation are indirect and are a consequence of impaired VDR action in the intestine which results in abnormal mineral ion homeostasis. This observation led us to focus our investigations on the pathophysiological basis for the development of rickets, which is an indirect consequence of VDR ablation. We demonstrated that the growth plate abnormalities in the VDR null mice are a consequence of hypophosphatemia, which leads to expansion of the late hypertrophic chondrocyte layer due to impaired apoptosis of these cells. Since hypophosphatemia leads to impaired terminal chondrocyte differentiation during postnatal life, studies are proposed to address the hypothesis that phosphate restriction also impairs chondrocyte differentiation during endochondral skeletal development and skeletal repair. Investigations will be performed using the ex vivo mouse metatarsal culture system and the in vivo femoral fracture repair models. These studies will identify developmental pathways that are interrupted by hypophosphatemia, an indirect consequence of VDR ablation. Analogous to humans with VDR mutations, VDR null mice have hair perinatally, but develop alopecia. Hair reconstitution assays and studies in transgenic mice demonstrate that VDR expression in keratinocytes is both necessary and sufficient for the prevention of alopecia, thus the effects of the VDR in this cell are direct. We have also demonstrated that the actions of the VDR required to prevent alopecia are ligand- independent, thus reflect novel molecular actions of this nuclear receptor. Investigations in this proposal will address the hypothesis that the VDR is a key regulator of the canonical Wnt signaling pathway in keratinocyte stem cells and that these ligand-independent actions of the VDR are required for cutaneous homeostasis. The goal of these studies is not to study keratinocyte biology per se, but rather to use this model system to characterize the molecular pathway and molecular partners involved in mediating these unique actions of the unliganded VDR. Thus, using the keratinocyte as a model, these studies will identify the novel molecular actions of the VDR which prevent the development of alopecia, a direct consequence of VDR ablation. [unreadable] [unreadable] [unreadable]